Myosin is found in all eukaryotic cells and appears to be involved in diverse cellular motile processes such as cytokinesis. Recently, this laboratory has isolated two different cDNAs for nonmuscle myosin heavy chains (MHCs) which are encoded by two different genes, in both chicken and human. We studied the expression of two MHC mRNAs in a number of chicken tissues and cultured fibroblasts using RNA blot analysis, and demonstrated a tissue and cell type-dependent expression for the two MHC mRNAs as well as changes in mRNA expression associated with the cell cycle in chicken embryo fibroblasts (Kawamoto and Adelstein, J. Cell Biol. 112: 915-924, 1991). Using human cDNA as a probe, we extended the above observations to a variety of mammalian cells, including human epidermoid carcinoma cells (A43 1), mouse myogenic cells (C2C 12), and rat aortic smooth muscle cells. The expression of the two MHC mRNAs varied depending on cell types and, moreover, the mRNA expression of the two MHCs can be differentially changed in various cell types in association with cell growth and differentiation. To understand the regulatory mechanisms controlling the expression of nonmuscle MHC genes, we initiated the cloning of a human nonmuscle MHC gene. The 5' untranslated region and the beginning of the coding region of a nonmuscle MHC mRNA were amplified by polymerase chain reaction following reverse transcription and subcloned. This 5' end cDNA has been used for the screening of a cosmid human genomic DNA library. A number of overlapping clones were isolated and partially characterized. 217 nucleotides of 5' untranslated region are encoded by two exons (the 2nd exon contains the initiation methionine codon) and a large intron (approximately 35 kbp) lies between them. The characterization of the putative promoter region is in progress.